This invention relates to apparatus for and methods of rapidly and sensitively detecting and monitoring chemical and biological materials.
Recent developments in the world political situation, exemplified by the demise of the Soviet Union, continued geopolitical pressures in the Middle East and Eastern Europe and the proliferation of terrorist activities throughout the world, have raised increased concerns about the use of chemical and biological warfare materials in local conflicts. The defense against chemical and biological warfare agents includes detection of potential threats, development and use of protective equipment, development of vaccination post-exposure prophylaxis measures and fabrication of structures providing barriers to the toxic agents which are suitable for decontamination procedures. Threat identification is imperative prior to engagement, during battle and after battle during decontamination procedures. In addition, chemical sensors for detecting chemical warfare materials are needed for treaty verification, demilitarization, environmental monitoring and characterization of materials acting as barriers to agent diffusion.
Existing methods of detection have proven inadequate. Existing methods for long-range threat identification, such as light detection and ranging (LIDAR), and for laboratory analysis of chemical warfare agents using gas chromatography to provide a chemical agent monitor (miniCAMS), light addressable potentiometric sensor (LAPS) or ion mobility sensor (IMS) technology, have proven slow and cumbersome to carry out. Needs exist for lightweight, high-sensitivity sensors having rapid response times.
Existing sensors have proven capable of meeting the requirements of several applications but no sensor has provided the combined sensitivity and speed of response needed for each application. Needs exist for field-usable chemical and biological sensors for the detection of vapor and liquid dispersed chemical warfare agents, toxins of biological origin and aerosol dispersed pathogenic microorganisms. Existing instrumentation used in identifying chemical warfare agents rely on ion mobility spectroscopy or gas chromatography for detection. The Advanced Chemical Agent Detection/Alarm System (ACADA) uses ion-mobility spectroscopy to achieve sensitivities to Sarin and Soman on the order of 1 mg/m3 (170 parts per billion (ppb)) in ten seconds and 0.1 mg/m3 (17 ppb) in 30 seconds. In addition to the system""s slow response and low sensitivity, the size and weight characteristics of the ACADA system (one cubic foot in volume and 25 pounds in weight) reduce the applicability of the system for distributed sensing or remote sensing applications. Sensors such as the miniCAMS system provide unparalleled sensitivity but require preconcentration times on the order of minutes. That response time is unsuitable for rapid detection of conditions that are immediately dangerous to life and health. Other existing methods use acoustic or optical/electrochemical methods of detection, such as surface acoustic wave (SAW)-based instruments and light addressable potentiometric sensors (LAPS). Neither method has proven effective in meeting the sensitivity and response times required. At best, the SAW instrument has demonstrated sensitivities to Sarin/Soman at 0.01 mg/m3 (1.7 ppb), but requires preconcentration times ranging from 2 minutes to 14 minutes. Needs exist for field-usable sensors that provide for highly-sensitive, rapid response measurements of the concentration of analytes in solution or in air.
Previous efforts indicate that polymers can be used to improve the sensitivity of SAW devices to a range of analytes and that polymer coatings are effective in enhancing the concentrations of analyte detected by optical probes. Polymer-coated waveguides have been used to detect nerve agent simulants, such as dimethyl-methyl-phosphonate (DMMP), and polymeric materials having affinity for the nerve agent and exhibiting a change in refractive index upon absorption of the agent have been identified. Floropolyol was found to have a partition coefficient for vapor phase DMMP between one million and ten million, indicating that the concentration of DMMP in the fluoropolyol was up to ten million times that in the vapor phase. Fluoropolyol is strongly acidic, which may improve sensitivity to strongly basic vapors such as the organophosphorus compounds. No sensors have been developed, however, that have short response times and high sensitivity, that are low cost and that are easily and safely incorporated into small, portable packages for deployment in rugged domains. Needs exist for optical chemical and biological sensors having those characteristics for use in a network of point detectors for multiple applications, including monitoring decontamination of military field structures, detecting chemical and biological agents in chemical treaty verification, assisting in reconnaissance of battlefield and depot perimeters, demilitarization exercises and monitoring breakthrough times associated with polymeric or other complex structural materials.
The far-visible and near-infrared (IR) spectral regions (600-1000 nm) are areas of low interference, where only several classes of molecules exhibit significant absorption and fluorescence. The use of near-IR labels in sensor design is becoming increasingly important due to the advent of semiconductor-based light sources and detectors and the reduced interference in the near-IR wavelength range. Heptamethine cyanine dyes have been shown effective in labeling nucleic acid materials. For biomolecule labeling and for analytes containing primary amino functional groups, isothiocynate derivatives of those cyanine dyes are the most suitable labels because they form stable thioureas. Needs exist for sensors for use in biochemical applications that use near-IR fluorophores.
Needs exist for low-cost sensors exhibiting rapid response at very low concentrations of chemical warfare agent and simulant materials. Needs also exist for laser-based sensors that can be used commercially in biomedical, industrial and environmental applications requiring on-site, rapid and sensitive chemical and biological analysis. In the field of biomedical testing, there is great need for point-of-care monitoring of physiological conditions and disease producing microorganisms. In the food industry, there is a need for sensors for product quality control and distributed process control applications. Needs for rapid, sensitive, small, portable and inexpensive sensors are also needed in the chemical and pharmaceutical processing industries, and in the area of pollution monitoring. Needs exist for sensors that can meet the sensitivity and time response requirements dictated by each industry and that are of a reasonable size and cost.
A highly-sensitive, rapid response fluorescent probe is based on the affinity of a polymer matrix for an analyte of interest.
The polymer/fluorophore probes of the present invention have the sensitivity and rapid response needed for detection of chemical agent and biological materials. Sensors using the probes provide sensitivity to Sarin at several hundred parts per trillion without the need for preconcentration steps. The lack of necessity for preconcentration allows vapors to be detected in one second or less. That is a notable advance over state-of-the-art detectors that require preconcentration steps, which in turn restrict response times to one minute or more.
Fluorescent dyes showing little or no sensitivity to an analyte of interest provide significant sensitivity to that same analyte of interest as long as a polymer having affinity for the analyte is employed as the matrix. A wide-range of near-infrared excitable fluorophores are used as sensitive probes for analytes not detectable when the fluorophores are outside the polymer matrix. The present sensors provide early warning of the presence of toxic chemicals, provide for on-line analysis of trace materials in chemical and biological processing operations and biomedical processing operations, and provide for effective biomedical and environmental monitoring.
Organic thin films having affinity for particular analytes and the immobilization of fluorescent dyes in those films to produce a sensitive apparatus and method for detecting chemical and biological materials. Perfluorinated, ionophoric polymeric materials produce self-assembled structures which, in the presence of semiconductor diode laser-excitable dyes, respond to targeted chemical or biological agents. The combination of thin polymer films with near-IR excitable dyes has advantages that include:
the diffusion and reaction zones of a sensor based on a fluorophore immobilized in a polymer matrix has reduced dimensions, thereby resulting in rapid sensor response;
organized thin polymer films are compatible with silicon, gallium arsenide, indium phosphide and silicon carbide semiconductor materials, thereby allowing optical integration into a small, rugged sensor package;
near-IR fluorophores are excited in a wavelength region where there are fewer interferences than observed with excitation in ultraviolet or visible regions of the spectrum and the compatibility of near-IR fluorophores with semiconductor diode laser excitation further contributes to improved signal-to-noise ratio with minimal package size;
since semiconductor diode lasers are inexpensive, rugged and suitable for field use, sensors developed based on fluorophores excitable by semiconductor diode lasers can be manufactured inexpensively and packaged for portability or for use in an array of point detectors.
In the present invention, thin films, such as poly(ethylene-maleic anhydride) (PEM), poly(vinyl pyridine) (PVP) and Nafion(copyright) films, are deposited on glass substrates and on polymer optical waveguides. The films are deposited with fluorescent materials sensitive to chemical agents and simulants. Preferably, the fluorescent material is selected from a wide range of near-IR fluorophores. The fluorophores in the thin polymer film are responsive to dimethyl methylphosphonate or nerve agent. Numerous polymer/fluorophore combinations are possible, each being capable of ameliorating present limitations in response times at low agent concentrations.
Several polymers are possible for use as the immobilization matrix for fluorescent dyes. When near-IR fluorescent dyes are incorporated in the matrix, sufficiently rapid response to nerve agent stimulants is provided to allow for real time detection of those materials. In one embodiment, Nafion(copyright) thin films with hexamethylindodicarbocyanine membrane potential-sensitive dyes are used in conjunction with semiconductor diode lasers to detect Sarin at concentrations as low as 500 parts per trillion (ppt). The sensor response time is approximately one second. The sensor is reversible to Sarin, in that removal of the chemical agent results in return of the original fluorescence level in about one second. The rapid response to Sarin represents an advance in the state-of-the-art of detection.
Chemical sensors using the thin polymer film/fluorescent material combination of the present invention provides for increased selectivity in the detection of chemical warfare and toxins of biological origin. The use of a semiconductor diode laser in the present sensor provides sensitivities compatible with single mode excitation and detection through interaction of the evanescent field of an optical waveguide with a chemically sensitive film deposited on the waveguide surface.
Sensors developed based on the polymer/fluorophore probes meet the requirements mandated for military applications. The present sensors detect nerve agent GB (Sarin) at concentrations less than 500 parts per trillion with a response and cleardown time of less than one second. No existing instruments provide response to that level in as short a time. The enhanced sensitivity of a membrane potential dye immobilized in a ionomeric, amphiphilic polymer film results from the unique charge environment in the vicinity of the dye. The most sensitive polymer/fluorophore probes respond irreversibly to a number of interferents but reversibly to chemical agents and simulants. Less sensitive polymer/fluorophore probes respond reversibly to both simulant and interferences.
The immobilization of fluorophores in polymeric matrices with enhanced affinity for an analyte of interest provides improved sensitivities over sensors fabricated with the same fluorophores in polymers with little or no affinity to the analyte of interest. That demonstrates the compatibility of surface acoustic wave sensor data with the selection of polymers suitable for optical detection. In particular, amphiphilic polymers combined with membrane potential sensitive dyes are particularly valuable for the detection of hazardous vapors. Similarly, polymer materials such as poly(ethylene-maleic anhydride) (PEM), poly(vinyl pyridine) (PVP) or Nafion(copyright), having a known affinity for dimethyl methylphosphonate (DMMP), when used in conjunction with fluorescent dyes, function as probes that are sensitive to DMMP. In one embodiment, reversible probes are produced using solvatochromic fluorophores immobilized in polymers having an affinity for an analyte of interest. Probes fabricated from PVP or PEM also respond reversibly to DMMP as well as to interferents such as ammonia.
The enhanced sensitivity of a membrane potential dye immobilized in an ionomeric, amphiphilic polymer, such as Nafion(copyright) film, to an analyte of interest, such as the nerve agent Sarin, results from the unique charge environment in the vicinity of the dye. Similar sensitivities are achieved using other matrices, including poly(ethylene-maleic anhydride) (PEM) or poly(vinyl pyridine) (PVP) on glass slides after doping with nile red or nile blue.
Diode laser excitation has advantages over excitation using conventional light sources. Instrument size is reduced by using diode lasers rather than conventional light sources. Semiconductor diode lasers are also noted for long operational lifetimes, in the range of 105 h.
Using a small sensor having the polymer/fluorescent probe, nerve gas is detected at 500 parts per trillion. Benzene, xylene and toluene are detectable at trace concentrations by using one of a wide range of semiconductor laser excitable dyes. Ammonia is also detectable at trace concentrations using the present invention. By employing differing dyes immobilized in differing polymer matrices, nearly any chemical can be identified and detected.
In addition to military applications, sensors having the polymer/fluorophore probes have potential biomedical, environmental, commercial and industrial applications. The present sensor can be used in environmental chemical and biological detection, including the monitoring of waste sites and ground water quality control, and in process evaluation and hazard analysis for quality control in the food processing, biotechnological and materials processing industries. The reduced cost and portability of the sensor offers advantages in process inspection, in point-of-care medical diagnosis and in environmental site monitoring.
A fluorescent probe apparatus for use in a sensor includes a polymer matrix and a dye immobilized in the matrix. The polymer matrix has an affinity for an analyte of interest and the dye has little or no sensitivity to the analyte of interest when excited by an excitation source in a free state but has significant sensitivity to the analyte of interest when excited by the excitation source when immobilized in the matrix. The dye is preferably a fluorescent dye that includes near-infrared fluorophores. The polymer matrix is preferably a poly(ethylene-maleic anhydride) matrix, a poly(vinyl pyridine) matrix, a poly(4-vinyl-phenol) bromonated matrix, or a Nafion(copyright) matrix.
In preferred applications, the analyte of interest is selected from the group consisting of dimethyl methylphosphonate, Sarin, benzene, xylene, toluene and ammonia.
In a preferred embodiment of the probe, the dye is a membrane potential sensitive dye and the polymer matrix is a micelle forming polymer matrix or a reverse micelle forming polymer matrix. In one embodiment, the membrane potential sensitive dye is 1,1xe2x80x23,3,3xe2x80x2,3xe2x80x2-hexamethylindotricarbocyanine iodide and the matrix is a Nafion(copyright) matrix.
In another embodiment, the dye is a fluorophore dye, the polymer matrix is a poly(isoprene/fluoro alcohol) matrix or a poly (ethyleneimine) matrix, and the analyte of interest is a hydrogen-bond forming material.
In another embodiment, the dye is nile red or nile blue, and the polymer matrix is a poly(ethylene-maleic anhydride) matrix or a poly(vinyl pyridine) matrix.
In a preferred embodiment of the probe, the dye is selected from the group consisting of nile blue 690 (5-amino-9-(diethylamino)-benzo[a]phenoxazin-7-ium perchlorate, quinaldine red, phenosafranin, rhodanile blue, Oxazine 750 (2,3,6,7-tetrahydro-5-(ethylimino)-1H,5H-benzo[a]phenoxazin-[8,9,10-ij]quinolizin perchlorate, Oxazine 170 (CAS Reg. 62669-60-7), (5,9-bis(ethylamino)-10-methyl-benzo[a]phenoxazin-7-ium perchlorate, brilliant crescyl blue, 3,3xe2x80x2-diethylthiatricarbocyanine iodide, 1,1xe2x80x23,3,3xe2x80x2,3xe2x80x2-hexamethylindodicarbocyanine iodide, IR-144 (CAS Reg. 54849-69-3) and methylene blue, and the polymer matrix is selected from the group consisting of a Nafion(copyright) matrix, a poly(ethylene maleate) matrix and a poly(vinyl pyridine) matrix.
The analyte of interest can be a live chemical agent. When a live agent is the analyte of interest, the dye is preferably nile blue perchlorate, 1,1xe2x80x23,3,3xe2x80x2,3xe2x80x2-hexamethylindodicarbocyanine iodide or Oxazine 750.
A method for chemical sensing includes the steps of immobilizing a dye in a polymer matrix to form a polymer/dye probe, depositing the probe on a substrate, exciting the probe positioned on the substrate, exposing the probe on the substrate to an analyte of interest, and detecting a change in the sensitivity of the dye immobilized in the polymer matrix of the probe. The method can further include the step of adding a strong base to the polymer matrix prior to or concurrently with the immobilizing step. Preferably, the strong base is sodium hydroxide or ammonium hydroxide.
In preferred embodiments, the depositing step is performed by spin coating or solution casting.
The present method can further include baking the probe/substrate combination after the depositing step. The baking step is preferably carried out in a nitrogen atmosphere.
The probe may be deposited on a glass slide substrate.
A highly-sensitive, rapid response sensor apparatus for detecting chemical and biological agents includes a dye/polymer probe, a waveguide connected to the probe, an excitation source for providing light to the waveguide and for exciting the dye, and a detector for detecting a response from the probe. The probe further includes a polymer matrix and a dye immobilized in the matrix. The polymer matrix has an affinity for an analyte of interest and the dye has little or no sensitivity to the analyte of interest when excited by the excitation source in a free state but has significant sensitivity to the analyte of interest when excited by the excitation source when immobilized in the matrix. The excitation source is preferably a semiconductor diode laser and the detector is a photomultiplier tube or an amplified photodiode.
In a preferred embodiment, the probe of the sensor includes a series of dye/polymer matrix probes that respond reversibly to the analyte of interest and to interferents.
In a preferred embodiment, the sensor further includes a modulator connected to the excitation source and to the detector for direct modulation of the excitation source. The modulator preferably includes a gain-phase analyzer connected to the detector and a bias tee connected to the analyzer and to the excitation source.
These and further and other objects and features of the invention are apparent in the disclosure, which includes the above and ongoing written specification, with the claims and the drawings.